Interpretive Summary: Research from a several scientific arenas, including agriculture, indicates that marked shifts in biological activity can result when the covalency of nitrogen in a biomolecule is chemically modified. A myriad of oxidants are well known to react with “free” and “combined” amino acids (i.e, peptides and proteins) to transform the covalency of amine nitrogen. Understanding oxidant-derived structural change to amine nitrogen in peptides and proteins has been obscured by challenges related to the analysis of the modified material. We developed a technique combining sample concentration by vacuum evaporation, enzymic polypeptide cleavage (i.e. proteolysis), and characterization and quantification of modified amino acid monomers using high pressure liquid chromatography mass spectrometry (HPLC-MS). We used Pronase E, an enzyme cocktail derived from Streptomyces griseus digestion, for proteolysis because it is hydrolytically active on many of the possible amino acid linkages. Using a multivariate statistical approach, we evaluated the efficacy of Pronase E liberation of amino acids from polypeptides across the range of pH, salinity, and NOM concentration conditions relevant to seawater, disinfected drinking waters, wastewaters, recreational waters, human blood, and plant tissue. The method was applied to characterize and quantify lysine nitrile, one of the predicted hypohalous acid-catalyzed transformation products of the primary amine moiety within the lysine side chains of polypeptides. The adaptation of this technique to studying protein modifications in plant-insect-microbe systems will undoubtedly shed new light on the molecular-level mechanisms that underpin their interaction.

Technical Abstract:
Characterizing the transformations of polypeptides is important across a broad range of scientific disciplines. As polypeptides are an important constituent of dissolved organic matter within seawater and freshwater, it is important to understand their fate. Oxidants formed in blood, as part of the immunological response, or applied to waters for disinfection react with polypeptides to form transformation products that may exert toxicity. An analytical method was developed to characterize and quantify modifications to the side chains of amino acid residues within polypeptides. For samples featuring protein concentrations <1 g/L, lyophilization achieved concentration without significant losses to the lyophilization vessel walls. Samples were digested enzymatically using Pronase E, a protease cocktail. Using liberation of a mass-labeled leucine monomer from an octapeptide spiked standard as a measure of complete digestion efficiency, a multivariate statistical analysis was conducted to evaluate the influence on digestion efficiency of Pronase E loadings, salinity, natural organic matter concentration, and pH across the range of conditions relevant to blood or concentrated seawaters and disinfected drinking/recreational waters. At Pronase E loadings of 10 mg, the analysis indicated that digestion efficiencies ranged from 25-55% over the range of conditions expected for typical drinking waters concentrated from 1 L to 10 mL. Lysine nitrile, one of the predicted transformation products of lysine residues within polypeptides, was destroyed by strong acid digestion, but preserved by the Pronase E enzymatic digestion. The analytical method was applied to triplicate 1 L samples of a chlorinated tap water and a chlorinated indoor pool water. For the tap water, the digestion efficiency was 47.2% (± 11.1% relative standard deviation), and the lysine nitrile concentration was 104.6 ng/L (± 6.8 ng/L standard deviation). For the pool water, the digestion efficiency was 23% (± 15% relative standard deviation). Although the occurrence of lysine nitrile was verified, the matrix prevented quantification.